Method for increasing mean survival times of transplants with LFA-1-specific antibodies

ABSTRACT

Mean survival times of a functional transplant of solid organs in a patient are increased by administering to the patient an initial dosing of a therapeutically effective amounts of a monoclonal antibody directed against the human LFA-1 molecule in the two hours after grafting and then administering daily dosing during a period of approximately nine days.

This is a U.S. national phase of PCT/FR94/00071, filed Jan. 21, 1994.

The present invention relates to the use of monoclonal antibodies forthe preparation of a medicinal product for preventing the rejection oftransplants of solid organs, to medicinal products comprising them andto a medical kit for effecting this prevention. Solid organs areunderstood to mean, in particular, the kidney, heart, lungs, liver andskin, an well as endocrine glands such as pituitary glands, thyroid andpancreas and cell suspensions extracted from these (for example isletsof Langerhans), and the like.

The last decade has seen an improvement in the results of organ, inparticular, kidney, transplantations, reflected in an improvement in thesurvival of the patients and of the grafts. Before 1981, the use ofazathioprine (AZA) and of prednisone (P) constituted the means ofprimary prophylactic immunosuppression in relation to renaltransplantation, despite a relatively low rate of survival of the graft.The appearance of cyclosporin A (CsA) enabled graft survival to besignificantly improved, approximately 90% surviving at one year (in“Annual Report of United Network for Organ Sharing” 1989, UNOS Eds.Richmond—J. M. Ceaka et al., The UNOS scientific renal transplantregistry, in “Clinical transplants” 1989: 1-8 UCLA Eds. Los Angeles),with a prolonged survival of the patients (approximately 95% in aone-year period), but with not-insignificant problems of toxicity an acounterpart. Polyclonal and monoclonal antilymphocyte antibodies havealso been included in protocols of prophylaxis and curative treatment ofrejection.

It has been possible to show low rates of early graft rejection by atwo-stage treatment comprising initially the use of polyclonalantilymphocyte antibodies and than of CsA as principal maintenanceimmunosuppressant (A. J. Richardson at al., Transplant Internat 1990; 3:26-31—B. G. Sommer et al., Transplantation 1987; 43: 85-90).

The introduction of monoclonal antibodies (mAb) having definedSpecificity has made it possible to envisage a more precise action interm of induction of immunosuppression and treatment of rejectionepisodes. At the present time, only the antibody OKT3 (anti-CD3 mAb) hasreceived a marketing authorization for the curative treatment of renaltransplant rejection episodes (Ortho-clone OKT®3, Product Information,Physicians' Desk Reference, 43rd edition (Medical Economics CompanyInc., N. J. Oradell, 1989, pages 1500-1501). However, only limitedevidence of its efficacy in preventing kidney transplant rejection (incombination with a chemical immunosuppression) is available. For themoment, no monoclonal antibody has been recognized for this indication.

The LFA-1 molecule (lymphocyte function-related molecule) in an integrinwhich belongs to the lymphocyte adhesion complex involved in thephenomena of cellular adhesion and intercellular communication and whichenhances, in particular, interactions between helper lymphocytes andtheir target cells. This family of products includes the Mac-1, LFA-1and Gp150,95 molecules which possess a common beta chain of 95 kD anddiffer from one another in their alpha chain. The LFA-1 or CD11a/CD18protein is a dimer of 180 kD present at the surface of bone marrow cells(leucocytic lines), T lymphocytes, NK cells, polymorphonuclearleucocytes and macrophages/monocytes. In vitro, monoclonal antibodiesdirected against LFA-1 inhibit most of the activities of T cells.

Anti-LFA-1 antibody has been used in children for the transplantation ofHLA-incompatible bone marrow (A. Fischer et al., Lancet 1986; ii,(8515): 1058-1061—N. Perez et al., Bone Marrow Transplant 1989; 4:379-384). Anti-LFA-1 antibodies have also been used in adult leukaemiapatients for preventing an HLA-induced T cell-depleted bone marrowtransplant rejection (D. Maraninchi et al., Bone Marrow Transplant 1989;4: 147-150). They have also been used to treat 10 patients displaying anacute graft-versus-host reaction resistant to steroid treatments (A. M.Stoppa et al., Transplant Int. 1991; 4: 3-7).

In relation to renal transplantation, anti-LFA-1 monoclonal antibodieshave been used in seven patients for treating episodes of acutetransplant rejection (B. Le Mauff et al., Transplantation 1991; 52 (2);291-296). The antibody used was the monoclonal antibody designated 25.3.Tolerance was good in the six patients who received more than oneadministration of this antibody. Infections were reported in twopatients. However, only one patient, probably the one who experiencedthe weakest episode of rejection, regained his renal function beforerejection, and a back-up treatment had to be instituted in five of them.In conclusion, this antibody was considered to be ineffective fortreating the acute rejection occurring in the course of kidneytransplantation. Use in the prevention of rejection has not beenstudied.

P. J. Berlin et al., Transplantation, Vol. 53, No. 4, 1992, BaltimoreMd., USA, have described some degree of efficacy of the administrationof anti-LFA-1 antibody for blocking the activity of T cells during therejection of a cutaneous allograft in monkeys, and a slight prolongationof the survival of the graft before rejection. The only graft survivingat three months corresponds to a treatment combining an anti-CD11 and ananti-CD2.

Monoclonal antibodies directed against ICAM-1, the natural ligand forLFA-1, have shown some results in kidney transplant rejection inprimates (B. A. Cosimi et al., Leukocyte Adhesion Molecules 1989: 274).The authors suggest that a treatment combining anti-ICAM-1 andanti-LFA-1 antibodies might be more effective (B. S. Cosimi et al., J.of Immunol., 1990, vol. 144, No. 12, 4604-4612).

M. Isobe, Proceedings of Int. Congress of Immunol. Budapest, Aug. 23-28,1992 (Ed. Hungarian Soc. for Immunol.) Springer, Berlin, 1992, 554,W-90-19, also suggests a synergistic effect of anti-ICAM-1 andanti-LFA-1 monoclonal antibodies in allograft tolerance in rodents.

The prior art, while recognizing some useful properties in anti-LFA-1antibodies, does not therefore suggest the use of these antibodies forpreventing the rejection of transplants of solid organs, except in theform of combinations with other antibodies also involved in cellularadhesion phenomena.

The Applicant has now found that it was possible to prevent therejection of transplants of solid organs, such as the kidney, by theadministration of monoclonal antibody directed against the human LFA-1(CD11a/CD18) molecule, this being achieved without combination withother antibodies or with cyclosporin A.

He also found that the efficacy of this use was greatly dependent on theimplementation of a novel administration protocol.

The subject of the present invention is hence the use of monoclonalantibodies directed against the human LFA-1 molecule for the preparationof a medicinal product intended for the primary treatment for preventingthe rejection of transplants of solid organs, in particular the kidney,in man. The monoclonal antibodies are preferably directed against thealpha chain of the LFA-1 molecule.

For the purposes of the present invention, antibodies are understood tomean human antibodies, non-human, for example murine, humanized,chimeric recombinant antibodies or other antibodies, as well as antibodyderivatives, fragments and the like. All these antibodies, including thederivatives, may be prepared by standard methods.

The monoclonal antibodies which are useful in the invention may becharacterized in that they react with:

T and B lymphocytes, monocytes, macrophages and polymorphonuclearleucocytes;

approximately 60% of thymocytes and prothymocytes;

T cell lines (for example MOLT-4, HPB-ALL and CEM lines);

the KG1 line, isolated from an acute myeloid leukaemia.

The monoclonal antibodies according to the invention have it as theirobjective to block the LFA-1 molecule and thus to reduce intercellularinteractions. The monoclonal antibody should inhibit adhesion and theeffector functions of T cells and NK cells.

Apart from the properties of binding, defined above, with the differentcell classes, the monoclonal antibodies according to the inventionadvantageously have all or part of the following properties in vitro:

inhibition of the mixed lymphocyte reaction and, partially, ofphytohaemagglutinin (PHA)-induced proliferation;

inhibition of T-dependent cytotoxicity and of NK cytotoxicity;

impairment of the power of adhesion of polymorphonuclear leucocytes toglass;

absence of reaction with the membrane of leucocytes of childrensuffering from a congenital immunodeficiency of the LFA-1 CR3 receptorand Gp 150,95 complex;

inhibition of the binding of complement component C3bi to its CR3receptor;

inhibition of the antibody-producing activity of antigen-specificT-helper cells;

partial inhibition of antigen-induced T cell proliferation;

absence of cellular proliferation and of TNF-αproduction;

decrease (at high concentration) of the proliferative responses inducedby the mitogens PMA, ConA, PWM and OKT3 in solution;

decrease in blast cells and in expression of the a chain of the IL-2receptor (CD25);

decrease in the proliferative responses of SEB-stimulated,monocyte-depleted cell suspensions or of mitomycin-treated alloreactiveB cells (MLR).

Preferably, the monoclonal antibody will be a mouse IgG₁, and inparticular antibody 25.3 (internal reference 25.3.1.19.3B7), alsodesignated 25.3.1 in Leucocyte Typing III, Oxford Sep. 21-26, 1986,deposited with the ECACC collection, PHLS Centre for AppliedMicrobiology & Research, Porton Down, Salisbury, Wilts, SP4 GJ6, U.K.under reference 92 120 309 on Dec. 3rd, 1992.

The monoclonal antibody intended for preventing the rejection oftransplants of solid organs in a recipient is preferably administered atthe rate of approximately 1 to 50 mg/day, and in particularapproximately 15 to 20 mg/day; preferably over a period ranging from 1to 30 d., and in particular from 3 to 14; preferably by perfusion, inparticular of the order of 30 min.

The subject of the invention is also a medicinal product comprising anactive principle at least one monoclonal antibody directed against thehuman LFA-1 molecule, in isotonic solution at a concentration ofapproximately 1 mg/ml in a suitable vehicle.

The subject of the invention is also a medicinal product comprising asactive principle at least one monoclonal antibody directed against thehuman LFA-1 molecule, in isotonic aqueous solution for perfusion, andstabilizing agents such as tris(hydroxymethyl)aminomethane and TWEEN 80.

Preferably, for a solution of one liter, the medicinal product insolution comprises from 0.1 to 10 g, and in particular approximately 1g, of monoclonal antibody, and from 0.1 to 10 g, and in particularapproximately 2.4 g, of tris(hydroxymethyl)aminomethane or the like, andfrom 0.1 to 10 of TWEEN 80, and in particular 2, per ten thousand, in anisotonic solution.

The medicinal products according to the invention preferably comprise amonoclonal antibody directed against the alpha chain of the LFA-1molecule, and more especially a monoclonal antibody as defined above.

The subject of the invention is also a kit intended for simultaneous orseparate administration or administration set over a period of time forthe purpose of preventing rejection of transplants of solid organs,comprising:

a medicinal product in which the active principle in at least oneantibody directed against the LFA-1 molecule, preferably a monoclonalantibody of the type described above,

and at least one other compound which is active in regard to preventingorgan transplant rejection, chosen in particular from the followingcompounds:

tacrolimus (FK-506),

azathioprine,

a steroid, such as prednisone or an equivalent corticosteroid,

cyclosporin A.

The subject of the invention is also a method for preventing therejection of transplants of solid organs such as the kidney, in which atleast one anti-LFA-1 monoclonal antibody as described above and, whereappropriate, at least one other agent chosen from those in the above kitis/are administered. Preferably, the monoclonal antibody is administeredat the rate of 1 to 50 mg/day, in particular approximately 20 mg/day;preferably by perfusion, in particular of the order of 30 min.

More especially, the subject of the invention is a method for preventingthe rejection of transplants of solid organs such as the kidney,characterized in that an initial dose of an anti-LFA-1 antibody isadministered shortly, in particular in the two hours, for example onehour, before re-establishment of the vascular continuity of theimplanted graft, and daily doses are then administered for approximately9 days.

The initial dose is preferably 30 mg (2×15 mg), and the following doses,in adults, are advantageously of the order of 15 mg, preferablyadministered by perfusion into a peripheral vein.

The dosage, expressed with reference to weight, is larger in children.

The method of prevention is noteworthy in that it can be advantageouslyimplemented without any other major medication, in particular withoutother monoclonal antibodies or anti-T gamma globulins, and withoutcyclosporin.

Advantageously, cyclosporin treatment may be instituted on the 9th or10th day at the time anti-LFA-1 treatment is stopped.

The method according to the invention can, moreover, be combined withcustomary corticoid and azothioprine treatment.

The invention will now be described in greater detail by means of anembodiment of the invention.

Monoclonal antibody 25.3 is harvested after culture in fermenters in aprotein-free medium. The weekly supernatants are concentrated and thenpurified by three chromatographic steps on Q SEPHAROSE FF, then SSEPHAROSE FF, then Q Sepharose FF.

Bottles of 5 ml of antibody 25.3 at a concentration of 1 mg/ml areprepared:

antibody 5 mg tris(hydroxymethyl)aminomethane 12.1 mg NaCl 43.5 mg TWEEN80 1 mg water for injections  qs 5 ml

Immunosuppressant treatments may be combined;

Cyclosporin A (SANDIMMUN®): at the rate of 8 to 10 mg/kg/dayadministered orally from the 9th day after transplantation; in one totwo administrations daily for an indeterminate period; or intravenouslyat the rate of 2 to 4 mg/kg/day.

Azathioprine (IMUREL®) at the rate of 2 mg/kg/d from the day oftransplantation, administered orally or intravenously, for a total doseof 25 mg; administration in a single portion; the daily dose can rangefrom 2 to 2.5 mg/kg.

Steroids: at the rate of 1 mg/kg/d of prednisone or an equivalentcorticosteroid, on days 1 to 5, administered orally; from the 6th day,the dose is decreased every 5 days by 10 mg, then complete withdrawal onthe 45th day.

Various treatments may be combined if necessary; for example:

oral antifungal such as amphotericin B (FUNGIZONE®) at the rate of 1.5mg/day for one month,

antibiotic such an sulphamethoxazole/trimethoprim (BACTRIM®) at the rateof 800 mg/day for 3 months,

treatment for fever: for example paracetamol administered orally at therate of 650 mg, as required,

treatment for pruritus using an antihistamine such as diphenhydramine atthe rate of 50 mg administered orally, as required.

Clinical Experience in Prophylaxis of Kidney Transplant Rejection

A non-randomized, open, single-centre phase II clinical trial is carriedout using fifteen patients who are to undergo a 1st renaltransplantation.

The first patient was included on May 25, 1992. All the patients weremonitored for three months post-transplantation.

The first five patients (patients 1 to 5) received 20 mg/day ofanti-LFA-1 antibody 25.3 for 10 days. In the light of the clinical andbiological results obtained in this first group, the next five patients(patients 6 to 10) received 15 mg/day. The last five patients received10 mg/day (patients 11 to 15).

Inclusion Criteria

First transplantation of kidney from cadaver, recipient between 18 and60 years of age, donor between 10 and 60 years of age, duration of coldischaemia <48 h and obtaining of a signed consent to participate.

Treatment Protocol Used

The antibody is perfused intravenously over a period of 30 minutes fromdays D1 (day of transplantation) to D10.

Relative to the standard triple therapy protocol customarily used in thecentre, the introduction of cyclosporin is delayed to the 9th daypost-transplantation in order to achieve the recommended residual levelson withdrawal of anti-LFA-1 (on D10). Azathioprine in administered at adose of 2 mg/kg/day from D1 to 5, then reduced by 10 mg every 5 daysfrom D6 and withdrawn on D45 post-transplantation.

Tolerance to Injections

The immediate tolerance to the injections is excellent: all the patientsreceived the whole of the treatment. No difference was observed betweenthe groups (20 or 15 mg) in terms of tolerance to the anti-LFA-1injection.

Biological tolerance: half of the patients became immunized against theantibody, but the immunization always occurred after the last day oftreatment (between D12 and D30).

Clinical course - Summary table Number of patients 5 5 5 Number ofpatients who could 5 5 4* be evaluated Anti-LFA-1 (mg/day) for 10 20 15  10  days Mean monitoring period (days) 90  90  90  Withdrawal oftreatment 0 0 0 Immunization on D15 3/5 0/5 2/4 on D30 3/5 2/5 1/4Rejection episodes Before D10 0 0 0 from D10 to D30 0 0 0 from D30 toD90 1 3 2 (patient No.) (1) (2) (2) Infectious episodes 0 3 4 (2 CMV₂ (2CMV 1 candidiasis) 1 candidiasis 1 pyelonephritis) Blood creatinine(micromol/l) mean ± s.e.m D1 545 ± 221 516 ± 137 617 ± 135 (n ≅ 5) D30148 ± 20  229 ± 109 140 ± 38  D90 167 ± 48  186 ± 118 133 ± 22  *1 caseof immediate arterial thrombosis

All the patients are alive and have a functional transplant at 3 monthspost-transplantation, with the exception of 1 patient in group 3 wholost his graft through an immediate arterial thrombosis unrelated to arejection phenomenon.

No rejection episode under anti-LFA-1 treatment was observed. Sixpatients displayed at least one rejection episode (between the first andthe second month). These rejection episodes were all resolved byconventional treatments.

The biological monitoring of the study entailed the study of severalparameters.

Measurement of circulating antibody levels was accomplished by animmunoenzymatic test detecting mouse immunoglobulins (Ig) in the serumby reference to a calibration curve of purified 25.3. Both 20 and 15 mggroups display high levels of circulating 25.3, reaching on average 12and 10 μg/ml at the end of the treatment. The difference between thefirst and second groups is not statistically significant, and 25.3 isstill detectable in both groups on D14, or even D20 in the case of somepatients. In the third group, the levels were markedly lower andscattered, but nevertheless significant four days after the lastinjection.

The antibody does not bring about any major modification of the numberof circulating cells; hence it does not have a depleting effect. On theother hand, the circulating cells, and especially lymphocytes andmonocytes, are coated with 25.3 to the point of saturation. In effect,when the patients' cells are studied by cytofluorometry, the labellingobtained with a fluorescein-coupled anti-mouse antibody is of the sameintensity with or without prior labelling in vitro with a saturatingamount of 25.3 (20 μg/mg). This saturation in still observed at D14 inthe case of 3/5 of the patients in group I and in the case of all thepatients in group II.

The presence of the circulating antibody and saturation of the cells isaccompanied by a functional blocking of the cells demonstrated inrespect of the adhesion capacities of the T lymphocytes. LFA-1/ICAM-1molecules are known to be involved in the heterotypic adhesion of Tlymphocytes to B lymphoblastoid target cells.

A test of adhesion between the T lymphocytes of the circulating bloodand a B line (Daudi) was hence developed. The lymphocytes are isolatedon a Ficoll gradient and then labelled with an anti-CD3 coupled tophycoerythrin (7 μg/ml), and the B line is labelled by indirectimmunofluorescence with an anti-Daudi monoclonal antibody and then afluorescein-coupled anti-mouse antibody. The cells, taken up in RPMImedium, are brought into contact in a 1:1 (mononuclear/Daudi cells)ratio, and incubated either at 4° C. or at 37° C. (with and withoutaddition of 25.3, 15 μg/ml). The reaction in stopped by adding 2 volumesof cold medium, and the cells are stored at 4° C. until analysed bycytometry within the 2 h which follow. In the analysis, those CD3+ cellswhich are doubly positive are measured. These doubly positive eventscorrespond to T-Daudi conjugates, as confirmed by the apparent increasein size (×2 or ×3) of the “doubly” labelled CD3+ cells. The number ofconjugates corresponds to the percentage of conjugates obtained at 37°C. minus the percentage obtained in the presence of 25.3 at aconcentration of 15 μg/ml.

The results obtained with the patients' cells are expressed in the formof an adhesion index, that is to say expressed with reference to thevalues obtained in the same experiment with cells of control subjects(healthy donors so an to normalize inter-trial variations).

In all the groups of patients, the adhesion index is at rock-bottomlevel during the treatment, and then rises gradually between D20 andD30. A single patient, in group I, has recovered a normal adhesion atD15 and displays, moreover, considerable immunization against 25.3, withthe presence of anti-idiotype antibodies. The recovery of the adhesioncapacities taken place, in fact, at the same time as the desaturation ofthe circulating cells.

The expression of the LFA-1 molecule on the patients' lymphocytes wasalso analyzed, during the treatment, by cytofluorometry. A decrease inthe intensity of fluorescence of CD11a visualized with 25.3, capable ofreaching 50%, could be observed in both groups. This modulation beginsas early as the first injection, but is greater between D10 and D15. Itis accompanied by an altogether parallel decrease in the expression ofCD18, that is to say of the β chain of the LFA-1 molecule.

Seven of the fourteen patients monitored displayed an immunization,without effects during the treatment. Only four patients showed, afterthe treatment, mouse IgG antibodies that were detectable beyond adilution to 1/300.

In short, 25.3 is an antibody lacking a depleting effect which, at thedoses used, saturates the LFA-1 sites present on the circulating cellsand thus brings about a blocking of LFA-1-dependent lymphocyte adhesioncapacities. The treatment may be accompanied by a modulation of thecellular expression of the LFA-1 molecule. It in capable of inducing anantibody response which is often small but sometimes (1 case/10 in thisstudy) large, after the treatment period, with the presence ofanti-idiotype antibodies.

What is claimed is:
 1. A method for increasing mean survival times of afunctional transplant of solid organs in a patient, comprisingadministering to the patient an initial dosing of a therapeuticallyeffective amount of a monoclonal antibody directed against the humanLFA-1 molecule in the two hours after grafting, and then administeringdaily dosing during a period of approximately 9 days.
 2. A methodaccording to claim 1, wherein said grafted solid organ is kidney.
 3. Amethod according to claim 1, wherein the monoclonal antibody is directedagainst the alpha chain of the LFA-1 molecule.
 4. A method according toclaims 1 or 3, wherein the monoclonal antibody reacts with: T and Blymphocytes, monocytes, macrophages and polymorphonuclear leucocytes;approximately 60% of the thymocytes and prothymocytes; T cell lines; theline KG1.
 5. A method according to claim 1, wherein the monoclonalantibody is a mouse IgG₁.
 6. A method according to claim 5, wherein themonocolonal antibody is the antibody 25.3 deposited with the ECACCcollection under reference 92 120
 309. 7. A method according to claim 1,wherein the monoclonal antibody is administered at the rate ofapproximately 1 to 50 mg of antibody/day.
 8. A method according to claim1, wherein the monoclonal antibody is administered at the rate ofapproximately 20 mg of antibody/day.
 9. A method according to claim 1,wherein the monoclonal antibody is administered by perfusion.
 10. Amethod according to claim 1, wherein the monoclonal antibody isadministered by perfusion of approximately 30 minutes.
 11. A methodaccording to claim 1, wherein the monoclonal antibody is administered ata loading dose before the revascularization of the graft, and a dailydose is then administered up to the 10^(th) day.
 12. A method accordingto claim 1, wherein the monocolonal antibody is administered at aloading dose of 30 mg before the revascularization of the graft, and adaily dose of 15 mg is then administered up to the 10th day.